POS1415 ENDOTHELIAL INFLAMMAGING AND MITOCHONDRIAL DYSFUNCTION IN THE ANTIPHOSPHOLIPID SYNDROME
Authors: C. Pericleous, R. Maughan, L. Kabir, A. Dabrowska, K. Paschalaki, D. Carling, A. Randi, D. Arachchillage, J. Mason
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Inflammageing, vascular ageing and mitochondrial dysfunction are important contributors to cardiovascular risk in the rheumatic diseases. In antiphospholipid syndrome (APS), endothelial dysfunction is underpinned by a thrombo-inflammatory state mediated by antiphospholipid antibodies (aPL). The contribution of endothelial senescence and metabolic disturbances is not known. <h3>Objectives</h3> To assess senescence, inflammation and metabolic function in <i>ex vivo</i> blood derived endothelial colony forming cells (ECFC), a novel source of patient endothelium. <h3>Methods</h3> ECFC were isolated from PBMC of 17 thrombotic APS and 11 healthy control (HC) donors, cultured in endothelial growth media containing 20% FBS. ECFC frequency, days from PBMC seeding to first colony appearance and days to initial passage (from P0 to P1) were similar between APS and HC. ECFC were phenotyped at P4 by flow cytometry (CD31+/CD144+/CD146+/CD14-/CD45-) and assayed at P4-P5. Proliferation was measured by fluorescent EdU incorporation. Senescence was determined by staining for senescence-associated β-galactosidase activity (SA-βgal) and nuclear DAPI, expressed as % senescent cells against total cell number. Mitochondrial function and glycolysis were assessed in live cells using extracellular flux assays (Seahorse). Molecular analysis was informed by RNA-seq (5 APS, 5 HC) and targets validated in the extended cohort at the mRNA and protein level. Data was analysed by non-parametric Mann-Whitney, ANOVA and Spearman's correlation tests. <h3>Results</h3> Impaired proliferative capacity and a greater proportion of senescent cells were observed in APS <i>vs</i> HC ECFC (median % senescent, 13.1% <i>vs</i> 7.9%) (p=0.02). RNA-seq analysis revealed 266 differentially expressed genes in APS ECFC (188 up, 78 down) primarily representing elevated inflammatory cytokines, chemokines, cell adhesion and fibrosis-remodelling molecules. Target validation at the mRNA level confirmed a hypoproliferative inflammatory phenotype in APS; elevated genes included <i>CDKN2A</i>/p16 (late senescence), <i>GADD45B</i> (growth arrest, DNA damage); <i>IFNA1</i> (cytokine), <i>CXCL10</i> (chemokine), <i>CD274</i>/PDL1 (exhaustion), <i>PTX3</i> (fibrosis-remodelling), <i>SERPINE1</i> (fibrinolysis-remodelling), <i>EDN1</i> (vasoconstrictor), all p&lt;0.05. A positive correlation was noted between senescent and inflammatory gene expression (e.g. <i>p16 vs CXCL10</i>, r=0.4, p=0.03). Upregulation of selected targets was confirmed at the protein level (e.g. p16 and PDL1 by flow cytometry, APS <i>vs</i> HC p=0.002 and p=0.03 respectively). Diminished mitochondrial respiration and ATP production (p&lt;0.01) coupled with elevated glycolysis (p=0.02) was seen in APS <i>vs</i> HC ECFC. Mitochondrial parameters positively correlated with proliferative capacity (r≥0.5, p&lt;0.01) and negatively correlated with senescence and inflammation (r≤-0.4, p&lt;0.05), indicating a relationship between endothelial health and mitochondrial function. Glycolysis negatively correlated with proliferation (r=-0.6, p&lt;0.01). Comparing ECFC from patients with previous cardiovascular events (CVE, n=9) <i>vs</i> those without (no-CVE, n=8) revealed more pronounced senescence and mitochondrial dysfunction in CVE, while greater loss of proliferative capacity was observed in no-CVE. Inflammatory markers varied between the two patient groups e.g. PTX3 was higher in CVE and CXCL10 in no-CVE. Collectively, these observations suggest different underlying biological processes in patients with severe thrombotic complications compared to those without. <h3>Conclusion</h3> We propose APS as a paradigm disease for immune-mediated endothelial ageing, defined by a hypoproliferative-senescent, inflamed and metabolically perturbed phenotype. Evidence for a more severe dysfunctional phenotype in ECFC from patients with CVE compared to no-CVE, agrees with published studies associating vascular ageing and mitochondrial damage with cardiovascular risk. Ongoing work is assessing the impact of <i>ex vivo</i> treatment with IgG aPL, cytokines and relevant drugs capable of modulating senescence and immunometabolic processes. <h3> Acknowledgements</h3> CP is funded by Versus Arthritis (21223) and the Imperial College-Wellcome Trust Institutional Strategic Support Fund. This work is dedicated to Professor Justin Mason who passed away in 2022. <h3>Disclosure of Interests</h3> None Declared.